Method of resolution and antiviral activity of 1,3-oxathiolane nuclesoside enantiomers

ABSTRACT

A process for the resolution of a racemic mixture of nucleoside enantiomers that includes the step of exposing the racemic mixture to an enzyme that preferentially catalyzes a reaction in one of the enantiomers. The nucleoside enantiomer (−)-2-hydroxymethyl-5-(5-flurocytosin-1-yl)-1,3-oxathiolane is an effective antiviral agent against HIV, HBV, and other viruses replicating in a similar manner.

This application is a continuation of application U.S. Ser. No.08/092,248, filed on Jul. 15, 1993, now abandoned, which is acontinuation of U.S. Ser. No. 07/736,089, filed on Jul. 26, 1991, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 07/659,760,filed on Feb. 22, 1991, now U.S. Pat. No. 5,210,085, which is acontinuation-in-part of U.S. Ser. No. 07/473,318, filed on Feb. 1, 1990,now U.S. Pat. No. 5,204,466.

U.S. Government has rights in this invention arising out of the partialfunding of work leading to this invention through the NationalInstitutes of Health Grant Nos. NIH 5-21935 and NIH AI-26055, as well asa Veteran's Administration Merit Review Award.

BACKGROUND OF THE INVENTION

This invention is in the area of biologically active nucleosides, andspecifically includes a method for the resolution of nucleosideenantiomers, including 1,3-oxathiolane nucleosides, and antiviralcompositions that include the enantiomerically enriched 1,3-oxathiolanenucleosides, (−) and(+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”).

This application is a continuation-in-part application of U.S. Ser. No.07/659,760, entitled “Method for the Synthesis, Compositions and Use of2′-Deoxy-5-Fluoro-3′-Thiacytidine and Related Compounds”, filed on Feb.22, 1991, by Dennis C. Liotta, Raymond Schinazi, and Woo-Baeg Choi, thatis a continuation-in-part application of U.S. Ser. No. 07/473,318,entitled “Method and Compositions for the Synthesis of BCH-189 andRelated Compounds,” filed on Feb. 1, 1990, by Dennis C. Liotta andWoo-Baeg Choi.

In 1981, acquired immune deficiency syndrome (AIDS) was identified as adisease that severely compromises the human immune system, and thatalmost without exception leads to death. In 1983, the etiological causeof AIDS was determined to be the human immunodeficiency virus (HIV). InDecember, 1990, the World Health Organization estimated that between 8and 10 million people worldwide were infected with HIV, and of thatnumber, between 1,000,000 and 1,400,000 were in the U.S.

In 1985, it was reported that the synthetic nucleoside3′-azido-3′-deoxythymidine (AZT) inhibits the replication of humanimmunodeficiency virus type 1. Since then, a number of other syntheticnucleosides, including 2′,3′-dideoxyinosine (DDI), 2′,3′-dideoxycytidine(DDC), 3′-fluoro-3′-deoxythymidine (FLT),2′,3′-dideoxy-2′,3′-didehydrothymidine (D4T), and3′-azido-2′,3′-dideoxyuridine (AZDU), have been proven to be effectiveagainst HIV. A number of other 2′,3′-dideoxynucleosides have beendemonstrated to inhibit the growth of a variety of other viruses invitro. It appears that, after cellular phosphorylation to the5′-triphosphate by cellular kinases, these synthetic nucleosides areincorporated into a growing strand of viral DNA, causing chaintermination due to the absence of the 3′-hydroxyl group.

In its triphosphate form, 3′-azido-3′-deoxythymidine is a potentinhibitor of HIV reverse transcriptase and has been approved by the FDAfor the treatment of AIDS. However, the benefits of AZT must be weighedagainst the severe adverse reactions of bone marrow suppression, nausea,myalgia, insomnia, severe headaches, anemia, peripheral neuropathy, andseizures. These adverse side effects often occur immediately aftertreatment begins, whereas a minimum of six weeks of therapy is necessaryto realize AZT's benefits. DDI, which has recently been approved by anFDA Committee for the treatment of AIDS, is also associated with anumber of side effects, including sporadic pancreatis and peripheralneuropathy.

Both DDC and D4T are potent inhibitors of HIV replication withactivities comparable (D4T) or superior (DDC) to AZT. However, both DDCand D4T are not efficiently converted to the corresponding5′-triphosphates in vivo. Both compounds are also toxic and can causeperipheral neuropathies in humans.

The success of various 2′,3′-dideoxynucleosides in inhibiting thereplication of HIV in vivo or in vitro has led a number of researchersto design and test nucleosides that substitute a heteroatom for thecarbon atom at the 3′-position of the nucleoside. Norbeck, et al.,disclose that (±)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine(referred to below as (±)-dioxolane-T) exhibits a modest activityagainst HIV (EC₅₀ of 20 μm in ATH8 cells), and is not toxic touninfected control cells at a concentration of 200 μm. TetrahedronLetters 30 (46), 6246, (1989).

European Patent Application Publication No. 0 382 526 filed by IAFBiochem International, Inc. discloses a number of substituted1,3-oxathiolanes with antiviral activity, and specifically reports thatthe racemic mixture (about the C4′-position) of the C1′-β isomer of2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as(±)-BCH-189) has approximately the same activity against HIV as AZT, andno cellular toxicity at therapeutic levels. (±)-BCH-189 has also beenfound to inhibit the replication of AZT-resistant, HIV isolates frompatients who have been treated with AZT for longer than 36 weeks.

To market a nucleoside for pharmaceutical purposes, it must not only beefficacious with low toxicity, it must also be cost effective tomanufacture. An extensive amount of research and development has beendirected toward new, low cost processes for large scale nucleosideproduction. 2′,3′-Dideoxynucleosides are currently prepared by either oftwo routes: derivatization of an intact nucleoside or condensation of aderivatized sugar moiety with a heterocyclic base. Although there arenumerous disadvantages associated with obtaining new nucleosideanalogues by modifying intact nucleosides, a major advantage of thisapproach is that the appropriate absolute stereochemistry has alreadybeen set by nature. However, this approach cannot be used in theproduction of nucleosides that contain either nonnaturally occurringbases or nonnaturally occurring carbohydrate moieties (and whichtherefore are not prepared from intact nucleosides), such as1,3-oxathiolane nucleosides and 1,3-dioxolane nucleosides.

When condensing a carbohydrate or carbohydrate-like moiety with aheterocyclic base to form a synthetic nucleoside, a nucleoside isproduced that has two chiral centers (at the C1′ and C4′-positions), andthus exists as a diasteromeric pair. Each diastereomer exists as a setof enantiomers. Therefore, the product is a mixture of four enantiomers.

It is often found that nucleosides with nonnaturally-occurringstereochemistry in either the C1′ or the C4′-positions are less activethan the same nucleoside with the stereochemistry as set by nature. Forexample, Carter, et al., have reported that the concentration of the(−)-enantiomer of carbovir (2′,3′-didehydro-2′,3′-dideoxyguanosine)required to reduce the reverse transcriptase activity by 50% (EC₅₀ ) is0.8 μM, whereas the EC₅₀ for the (+)-enantiomer of carbovir is greaterthan 60 μM. Antimicrobial Agents and Chemotherapy, 34:6, 1297-1300 (June1990).

U.S. Ser. No. 07/659,760 discloses that 1,3-oxathiolane and1,3-dioxolane nucleosides can be prepared with high diastereoselectivity(high percentage of nucleoside with a β configuration of the bond fromthe C1′-carbon to the heterocyclic base) by careful selection of theLewis acid used in the condensation process. It was discovered thatcondensation of a 1,3-oxathiolane nucleoside with a base occurs withalmost complete β-stereospecificity when stannic chloride is used as thecondensation catalyst, and condensation of 1,3-dioxolane with a baseoccurs with almost complete β-stereospecificity when variouschlorotitanium catalysts are employed. Other Lewis acids provide low (orno) C1′-β selectivity or simply fail to catalyze the reactions.

There remains a strong need to provide a cost effective, commerciallyviable method to obtain β-stereospecificity of synthetic nucleosidesprepared by condensing a carbohydrate-like moiety with a base. This isimportant because it is likely that many synthetic nucleoside inhibitorsof viral replication now emerging from academic and commerciallaboratories will require resolution. An economical and facile methodfor resolving racemic mixtures of nucleosides would greatly facilitateantiviral research and ultimately, commercial manufacture. Further,resolution of racemic mixtures of nucleosides may provide a route toincrease the activity of synthetic nucleosides by eliminating orminimizing the undesired enantiomer.

Therefore, it is an object of the present invention to provide a methodfor the resolution of racemic mixtures of nucleosides.

It is another object of the present invention to provideenantiomerically enriched 1,3-oxathiolane nucleosides.

It is still another object of the present invention to provideenantiomerically enriched 1,3-oxathiolane nucleosides with significantantiviral activity and low toxicity.

SUMMARY OF THE INVENTION

A process for the resolution of a racemic mixture of nucleosideenantiomers or their derivatives is disclosed that includes the step ofexposing the racemic mixture to an enzyme that preferentially catalyzesa reaction in one of the enantiomers. The process can be used to resolvea wide variety of nucleosides, including pyrimidine and purinenucleosides that are optionally substituted in the carbohydrate moietyor base moiety. The process can also be used to resolve nucleosidederivatives that contain additional heteroatoms in the carbohydratemoiety, for example, FTC and BCH-189. The resolution of nucleosides canbe performed on large scale at moderate cost.

It has been discovered that the nucleoside enantiomer (−)2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”)exhibits significant activity against HIV (EC₅₀ of 0.0077 to 0.02 μM),HBV (hepatitis B virus), and other viruses replicating in a similarmanner. The (+)-enantiomer of FTC is also active against HIV (EC₅₀ of0.28-0.84 μM).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the chemical structure of2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”).

FIG. 2 is an illustration of a method for the preparation ofenantiomerically enriched cytidine and uridine 1,3-oxathiolanenucleosides.

FIG. 3 is a graph indicating the progress of lipase-catalyzed hydrolysisof the 5′-butyryl ester of FTC over time using the enzymes PS800 (-opensquare-) and PLE (-open circle with dot-).

FIG. 4 is a graph of the effect of concentration (μM) of racemic andenantiomerically enriched FTC (prepared by the method of Example 3)versus the percent inhibition of human PBM cells infected with HIV-1.((-darkened circle-, (±)-FTC), (-circle-, (−)-FTC), (-darkenedsquare-,(+)-FTC).

FIG. 5 is a graph of the effect of concentration (μM) of racemic andenantiomerically enriched FTC (prepared by method of Example 2) on thepercent inhibition of human PBM cells infected with HIV-1. ((-darkenedcircle-, (±)-FTC), (-circle -,(−)-FTC), (-darkened square-,(+)-FTC).

FIG. 6 is a graph of the effect of concentration (μM) of racemic andenantiomerically enriched BCH-189 (prepared by the method of Example 3)on the percent inhibition of human PBM cells infected with HIV-1.((-darkened circle-, (±)-BCH-189), (-circle-, (−)-BCH-189), (-darkenedsquare-,(+)-BCH-189).

FIG. 7 is a graph of the uptake of tritiated (±)-FTC in human PBM cells(average of two determinations) in time (hours) versus pmol/10⁶ cells.

FIG. 8 is a graph of the egress of radiolabeled (±)-FTC from human PBMcells, measured in hours versus pmol/10⁶ cells ((-darkened square-),(±)-FTC; (-///-), (±)-FTC monophosphate; (-// // //-), (±)-FTCdiphosphate; and (-/// ///-), (±)-FTC triphosphate).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “enantiomerically enriched nucleoside” refersto a nucleoside composition that includes at least 95% of a singleenantiomer of that nucleoside.

As used herein, the term BCH-189 refers to2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane.

As used herein, the term FTC refers to2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.

As used herein, the term “preferential enzyme catalysis” refers tocatalysis by an enzyme that favors one substrate over another.

I. Resolution of Racemic Mixtures of Nucleosides During the Hydrolysis

A method is provided for the resolution of racemic mixtures ofnucleoside enantiomers. The method involves the use of an enzyme thatpreferentially catalyzes a reaction of one enantiomer in a racemicmixture. The reacted enantiomer is separated from the unreactedenantiomer on the basis of the new difference in physical structure.Given the disclosure herein, one of skill in the art will be able tochoose an enzyme that is selective for the nucleoside enantiomer ofchoice (or selective for the undesired enantiomer, as a method ofeliminating it), by selecting of one of the enzymes discussed below orby systematic evaluation of other known enzymes. Given this disclosure,one of skill in the art will also know how to modify the substrate asnecessary to attain the desired resolution. Through the use of eitherchiral NMR shift reagents, polarimetry, or chiral HPLC, the opticalenrichment of the recovered ester can be determined.

The following examples further illustrate the use of enzymes to resolveracemic mixtures of enantiomers. Other known methods of resolution ofracemic mixtures can be used in combination with the method ofresolution disclosed herein. All of these modifications are consideredwithin the scope of the invention.

Resolution Based on Hydrolysis of C5′-Nucleoside Esters

In one embodiment, the method includes reacting the C5′-hydroxyl groupof a mixture of nucleoside racemates with an acyl compound to formC5′-esters in which the nucleoside is in the “carbinol” end of theester. The racemic mixture of nucleoside C5′-esters is then treated withan enzyme that preferentially cleaves, or hydrolyses, one of theenantiomers and not the other.

An advantage of this method is that it can be used to resolve a widevariety of nucleosides, including pyrimidine and purine nucleosides thatare optionally substituted in the carbohydrate moiety or base moiety.The method can also be used to resolve nucleoside derivatives thatcontain additional heteroatoms in the carbohydrate moiety, for example,FTC and BCH-189. The broad applicability of this method resides in parton the fact that although the carbinol portion of the ester plays a rolein the ability of an enzyme to differentiate enantiomers, the majorrecognition site for these enzymes is in the carboxylic acid portion ofthe ester. Further, one may be able to successfully extrapolate theresults of one enzyme/substrate study to another, seemingly-differentsystem, provided that the carboxylic acid portions of the two substratesare the same or substantially similar.

Another advantage of this method is that it is regioselective. Enzymesthat hydrolyse esters typically do not catalyze extraneous reactions inother portions of the molecule. For example, the enzyme lipase catalysesthe hydrolysis of the ester of 2-hydroxymethyl-5-oxo-1,3-oxathiolanewithout hydrolysing the internal lactone. This contrasts markedly with“chemical” approaches to ester hydrolysis.

Still another advantage of this method is that the separation of theunhydrolysed enantiomer and the hydrolysed enantiomer from the reactionmixture is quite simple. The unhydrolysed enantiomer is more lipophilicthan the hydrolysed enantiomer and can be efficiently recovered bysimple extraction with one of a wide variety of nonpolar organicsolvents or solvent mixtures, including hexane and hexane/ether. Theless lipophilic, more polar hydrolysed enantiomer can then be obtainedby extraction with a more polar organic solvent, for example, ethylacetate, or by lyophilization, followed by extraction with ethanol ormethanol. Alcohol should be avoided during the hydrolysis because it candenature enzymes under certain conditions.

Enzymes and Substrates

With the proper matching of enzyme and substrate, conditions can beestablished for the isolation of either nucleoside enantiomer. Thedesired enantiomer can be isolated by treatment of the racemic mixturewith an enzyme that hydrolyses the desired enantiomer (followed byextraction of the polar hydrolysate with a polar solvent) or bytreatment with an enzyme that hydrolyses the undesired, enantiomer(followed by removal of the undesired enantiomer with a nonpolarsolvent).

Enzymes that catalyze the hydrolysis of esters include esterases, forexample pig liver esterase, lipases, including porcine pancreatic lipaseand Amano PS-800 lipase, substillisin, and α-chymotrypsin.

The most effective acyl group to be used to esterify the C5′-position ofthe nucleoside can be determined without undue experimentation byevaluation of a number of homologs using the selected enzyme system. Forexample, when 1,3-oxathiolane nucleosides are esterified with butyricacid, resolutions with both pig liver esterase and Amano PS-800 proceedwith high enantioselectivity (94-100 enantiomeric excess) and oppositeselectivity. Non-limiting examples of other acyl groups that can beevaluated for use with a particular nucleoside enantiomeric mixture andparticular enzyme include alkyl carboxylic acids and substituted alkylcarboxylic acids, including acetic acid, propionic acid, butyric acid,and pentanoic acid. With certain enzymes, it may be preferred to use anacyl compound that is significantly electron-withdrawing to facilitatehydrolysis by weakening the ester bond. Examples of electron-withdrawingacyl groups include α-haloesters such as 2-chloropropionic acid,2-chlorobutyric acid, and 2-chloropentanoic acid. α-Haloesters areexcellent substrates for lipases.

Resolution Conditions

The enzymatic hydrolyses are typically carried out with a catalyticamount of the enzyme in an aqueous buffer that has a pH that is close tothe optimum pH for the enzyme in question. As the reaction proceeds, thepH drops as a result of liberated carboxylic acid. Aqueous base shouldbe added to maintain the pH near the optimum value for the enzyme. Theprogress of the reaction can be easily determined by monitoring thechange in pH and the amount of base needed to maintain pH. Thehydrophobic ester (the unhydrolysed enantiomer) and the more polaralcohol (the hydrolysed enantiomer) can be sequentially and selectivelyextracted from the solution by the judicious choice of organic solvents.Alternatively, the material to be resolved can be passed through acolumn that contains the enzyme immobilized on a solid support.

Enzymatic hydrolyses performed under heterogeneous conditions can sufferfrom poor reproducibility. Therefore, it is preferred that thehydrolysis be performed under homogeneous conditions. Alcohol solventsare not preferred because they can denature the enzymes. Homogeneity canbe achieved through the use of non-ionic surfactants, such as TritonX-100. However, addition of these surfactants not only assists indissolving the starting material, they also enhance the aqueoussolubility of the product. Therefore, although the enzymatic reactioncan proceed more effectively with the addition of a non-ionic surfactantthan under heterogeneous conditions, the isolation of both the recoveredstarting material and the product can be made more difficult. Theproduct can be isolated with appropriate chromatographic and chemical(e.g., selective salt formation) techniques. Diacylated nucleosides canbe used but are often quite lipophilic and hard to dissolve in themedium used.

EXAMPLE 1 Enantioselective Lipase-Catalyzed Hydrolysis of FTC Esters

A number of 5′-O-acyl derivatives of FTC were prepared by selectiveO-acylation of the N-hydrochloride salt (see Table 1) of (±)-FTC. Theefficiency of the hydrolysis of the derivatives by lipases wasinvestigated. As shown in Table 1, pig liver esterase (PLE) exhibits ahigh level of selectivity for the hydrolysis of the ester of the(+)-enantiomer of FTC. In contrast, PS-800 hydrolyses the ester of the(−)-enantiomer of FTC preferentially. The rate of the hydrolysis wasalso found to be dependent on the nature of the acyl group; the acetylderivative was significantly slower than the butyryl derivative. It hasnow been discovered that the rate of the hydrolysis of the propionicacid ester of FTC is even faster than that observed for the butyratederivative. % Recovery and % ee were both determined using HPLC.Although the enantioselectivity is excellent when employing PLE(typically 97% e.e. or higher), additional enrichment can beaccomplished by sequential enzymatic hydrolysis reactions in which theenantiomerically-enriched butyrate from a PLE-catalyzed hydrolysis issubjected to enzymatic hydrolysis by PS-800.

TABLE 1 Comparison of Effect of Ester on Enzyme Hydrolysis. substrate %recovery % e.e. (s.m.) FTC Esters with PLE: acetate 32.68 N.D.propionate 39.87 N.D. butyrate 48.00 98 butyrate 45.71 98.6 FTC Esterswith PS800: acetate 73.17 N.D. propionate 52.67 N.D. butyrate 58.34 N.D.valerate 41.50 94

EXAMPLE 2 Procedure for the Preparation of (+)- and (−)-FTC viaEnantioselective, Lipase-Catalyzed Hydrolysis of FTC Butyrate

The 5′-O-butyrate of (±)-FTC (1) (0.47 mmol, 149 mg) was dissolved in 16mL of a solution of 4:1 pH 8 buffer:CH₃CN. The clear solution wasstirred and treated with 26 mg of pig liver esterase (PLE-A). Theprogress of the reaction was monitored by HPLC (FIG. 3). After 20 hours(52% conversion), the reaction mixture was extracted with 2×80 mL ofCHCl₃ and 80 mL of ethyl acetate. The organic layer extracts werecombined, dried over anhydrous MgSO₄, filtered, and concentrated byrotary evaporation. The resulting residue was eluted on 2×1000 m pTLCplates using ethyl acetate as eluant (double elution) to give, afterisolation, 53 mg (36% based on starting material) of FTC butyrate whichwas determined to have 98% enantiomeric excess (e.e.) by HPLC analysis.The enantiomerically-enriched butyrate was then treated with 1.6 mL ofmethanol followed by 0.38 mmol (20 mg) of sodium methoxide. Theresulting mixture was stirred at room temperature, and the progress ofthe reaction was monitored by HPLC. The reaction was completed within 30minutes. The solvent was removed by rotary evaporation to give a crudewhite solid (76 mg) that was eluted on a 1000 m pTLC using 5:1 ethylacetate:ethanol. (−)-FTC was isolated as a white solid (33 mg; 82%yield). HPLC analysis of the FTC as its 5′-O-acetate derivative showed97% e.e.; [α](²⁰,_(D)) −120.5°(c =0.88; abs. ethanol).

Similarly, 1.2 mmol (375 mg) of the 5′-O-butyrate of (+)-FTC wasdissolved in 40 mL of 4:1 pH 8 buffer-CH₃CN. The clear solution wasstirred and treated with 58 mg of pig liver esterase (PLE-A). Theprogress of the reaction was monitored by HPLC. After 90 minutes (38%conversion), the reaction mixture was added to 150 mL of CHCl. Thelayers were separated and the aqueous layer lyophilized to removesolvent. The white residue from the lyophilization was extracted with3×10 mL of absolute ethanol. The extracts were filtered, combined, andconcentrated in vacuo to yield 179 mg of crude oil. The crude materialwas eluted on a 45×30 mm column of silica gel using 3×75 mL of ethylacetate followed by 5:1 ethyl acetate-ethanol. (+)-FTC (a) was isolatedas a white solid (109 mg; 37% based on starting butyrate). HPLC analysisof the (+)-FTC as its 5′-O-acetate derivative showed 97.4% e.e.;[a]O(²⁰,_(D)) +113.4° (c=2.53; absolute ethanol)

A similar reaction was performed using 0.12 mmol (37 mg) of the5′-O-butyrate of FTC and 7 mg of PS-800 in 4.0 mL of 4:1 pH 8buffer:CH₃CN. The reaction was considerably slower than that with PLE-Aand required 74 hours for 59% conversion. The recovered butyrate (11.4mg; 31% of the initial amount) was found to be 94% e.e. by HPLC.

Resolution of Nucleoside Enantiomers with Cytidine-DeoxycytidineDeaminase

In an alternative embodiment, cytidine-deoxycytidine deaminase is usedto resolve racemic mixtures of2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane and its derivatives,including 2-hydroxymethyl-5-(5-fluoro-cytosin-1-yl)-1,3-oxathiolane. Theenzyme catalyses the deamination of the cytosine moiety to a uridine. Ithas been discovered that one of the enantiomers of 1,3-oxathiolanenucleosides is a preferred substrate for cytidine-deoxycytidinedeaminase. The enantiomer that is not converted to a uridine (andtherefore is still basic) is extracted from the solution with an acidicsolution. Care should be taken to avoid strong acidic solutions (pHbelow 3.0), that may cleave the oxathiolane ring.

Cytidine-deoxycytidine deaminase can be isolated from rat liver or humanliver, or expressed from recombinant sequences in procaryotic systemsuch as in E. coli.

The method of resolution of cytidine nucleoside enantiomers usingcytidine-deoxycytidine deaminase can be used as the sole method ofresolution or can be used in combination with other methods ofresolution, including resolution by enzymatic hydrolysis of5′-O-nucleoside esters as described above.

Combination of Enzymatic Resolution with Classical Resolution Methods

The process described above for resolving racemic mixtures of nucleosideenantiomers can be combined with other classical methods of enantiomericresolution to increase the optical purity of the final product.

Classical methods of resolution include a variety of physical andchemical techniques. Often the simplest and most efficient technique isrecrystallization, based on the principle that racemates are often moresoluble than the corresponding individual enantiomers. Recrystallizationcan be performed at any stage, including on the acylated compounds andor the final enantiomeric product. If successful, this simple approachrepresents a method of choice.

When recrystallization fails to provide material of acceptable opticalpurity, other methods can be evaluated. If the nucleoside is basic (forexample, a cytidine) one can use chiral acids that form diastereomericmixtures that may possess significantly different solubility properties.Nonlimiting examples of chiral acids include malic acid, mandelic acid,dibenzoyl tartaric acid, 3-bromocamphor-8-sulfonic acid,10-camphorsulfonic acid, and di-p-toluoyltartaric acid. Similarly,acylation of the free hydroxyl group with a chiral acid derivative alsoresults in the formation of diastereomeric mixtures whose physicalproperties may differ sufficiently to permit separation.

Small amounts of enantiomerically enriched nucleosides can be obtainedor purified by passing the racemic mixture through an HPLC column thathas been designed for chiral separations, including cyclodextrin bondedcolumns marketed by Rainin Corporation.

EXAMPLE 3 Separation of Racemic Mixtures of Nucleosides by HPLC

The resolutions of the C4′-enantiomers of (±)-BCH-189 and (±)-FTC wereperformed using a chiral cyclodextrin bonded (cyclobond AC-I) columnobtained from Rainin Corporation (Woburn, Mass.). The conditions were asfollows: Isocratic 0.5% methanol in water; flow rate 1 ml/min., UVdetection at 262 nm. HPLC grade methanol was obtained from J. T. Baker(Phillipsburg, N.J.). The racemic mixtures were injected and fractionswere collected. Fractions containing each of the enantiomers werepooled, frozen, and then lyophilized. The compounds were characterizedby UV spectroscopy and by their retention times on HPLC. In general, the(−)-enantiomers have lower retention times than the (+)-enantiomers (seeJ. Liquid Chromatography 7:353-376, 1984). The concentrations of thecompounds were determined by UV spectroscopy, using a stock solution ofknown concentration (15 μM) prepared in water for biological evaluation.The retention times for the separated enantiomers are provided in Table2.

TABLE 2 Retention Times of Enantiomers of BCH-189 and FTC Compound R_(t)(min) (−)-BCH-189 9.0 (+)-BCH-189 10.0 (−)-FTC 8.3 (+)-FTC 8.7

II. Antiviral Activity of2-Hydroxymethyl-5-(5-Fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”)

It is often desirable to screen a number of racemic mixtures ofnucleosides as a preliminary step to determine which warrant furtherresolution into enantiomerically enriched components and evaluation ofantiviral activity. The ability of nucleosides to inhibit HIV can bemeasured by various experimental techniques. The technique used herein,and described in detail below, measures the inhibition of viralreplication in phytohemagglutinin (PHA) stimulated human peripheralblood mononuclear (PBM) cells infected with HIV-1 (strain LAV). Theamount of virus produced is determined by measuring the virus-codedreverse transcriptase enzyme. The amount of enzyme produced isproportional to the amount of virus produced. Table 4 provides the EC₅₀values (concentration of nucleoside that inhibits the replication of thevirus by 50% in PBM cells) and IC₅₀ values (concentration of nucleosidethat inhibits 50% of the growth of mitogen-stimulated uninfected humanPBM cells) of a number of (±)-1,3-oxathiolane and nucleosides.

EXAMPLE 4 Anti-HIV activity of (±)-1,3-Oxathiolane Nucleosides

A. Three-day-old phytohemagglutinin-stimulated PBM cells (10⁶ cells/ml)from hepatitis B and HIV-1 seronegative healthy donors were infectedwith HIV-1 (strain LAV) at a concentration of about 100 times the 50%tissue culture infectious dose (TICD 50) per ml and cultured in thepresence and absence of various concentrations of antiviral compounds.

B. Approximately one hour after infection, the medium, with the compoundto be tested (2 times the final concentration in medium) or withoutcompound, was added to the flasks (5 ml; final volume 10 ml). AZT wasused as a positive control.

C. The cells were exposed to the virus (about 2×10⁵ dpm/ml, asdetermined by reverse transcriptase assay) and then placed in a CO₂incubator. HIV-1 (strain LAV) was obtained from the Center for DiseaseControl, Atlanta, Ga. The methods used for culturing the PBM cells,harvesting the virus and determining the reverse transcriptase activitywere those described by McDougal et al. (J. Immun. Meth. 76, 171-183,1985) and Spira et al. (J. Clin. Meth. 25, 97-99, 1987), except thatfungizone was not included in the medium (see Schinazi, et al.,Antimicrob. Agents Chemother. 32, 1784-1787 (1988); Id., 34:1061-1067(1990)).

D. On day 6, the cells and supernatant were transferred to a 15 ml tubeand centrifuged at about 900 g for 10 minutes. Five ml of supernatantwere removed and the virus was concentrated by centrifugation at 40,000rpm for 30 minutes (Beckman 70.1 Ti rotor). The solubilized virus pelletwas processed for determination of the levels of reverse transcriptase.Results are expressed in dpm/ml of sampled supernatant. Virus fromsmaller volumes of supernatant (1 ml) can also be concentrated bycentrifugation prior to solubilization and determination of reversetranscriptase levels.

The median effective (EC₅₀) concentration was determined by the medianeffect method (Antimicrob. Agents Chemother. 30, 491-498 (1986).Briefly, the percent inhibition of virus, as determined frommeasurements of reverse transcriptase, is plotted versus the micromolarconcentration of compound. The EC₅₀ is the concentration of compound atwhich there is a 50% inhibition of viral growth.

E. Mitogen stimulated uninfected human PBM cells (3.8×10⁵ cells/ml) werecultured in the presence and absence of drug under similar conditions asthose used for the antiviral assay described above. The cells werecounted after 6 days using a hemacytometer and the trypan blue exclusionmethod, as described by Schinazi et al., Antimicrobial Agents andChemotherapy, 22(3), 499 (1982). The IC₅₀ is the concentration ofcompound which inhibits 50% of normal cell growth.

TABLE 3 EC₅₀ and IC₅₀ of Various Analogues of 1,3-OxathiolaneNucleosides in human PBM cells. Antiviral Cytotoxicity Code X or Y REC₅₀, μM IC₅₀, μM DLS-009 X = O H >100 >100 DLS-010 X = O Me 64.4 >100DLS-027 X = O F >100 >100 DLS-028 X = O Cl 60.8 >100 DLS-044 X = OBr >100 >100 DLS-029 X = O I >100 >100 DLS-020 Y = NH₂ H 0.02 >100DLS-011 Y = NH₂ Me >10 >100 DLS-022 Y = NH₂ F 0.011 >100 DLS-023 Y = NH₂Cl 38.7 >100 DLS-021 Y = NH₂ Br 77.4 >100 DLS-026 Y = NH₂ I 0.72 >100DLS-058(−) Y = NH₂ F 0.0077 >100 DLS-059(+) Y = NH2 F 0.84 >100 DLS-053Y = NH₂ CF₃ 60.7 >100

As indicated in Table 3, in general, the substituted cytosine1,3-oxathiolane nucleosides are more active than the correspondinguracil nucleosides. One of the compounds, (±)-FTC, (referred to as“DLS-022”, compound 8) not only exhibited exceptional activity(approximately 10 nM in PBM cells), but also quite low toxicity (>100 μMin PBM, Vero and CEM cells). This activity compares quite favorably with2′,3′-dideoxyadenosine (DDA, EC₅₀=0.91 μM),3′-azido-2′,3′-dideoxyuridine (AZDU, EC₅₀=0.18-0.46 μM),3′-dideoxythymidine (DDT, EC₅₀=0.17 μM), and dideoxycytidine (DDC,EC₅₀=0.011 μM).

The IC₅₀ of (±)-FTC was measured as over 100 μM, indicating that thecompound was not toxic in uninfected PBM cells evaluated up to 100 μM.

EXAMPLE 5 Antiviral Activity of the Enantiomers of FTC Resolved by HPLC

The enantiomers of FTC were isolated by the method of Example 3, and theantiviral activity evaluated by the method of Example 4. The results areprovided in Table 4, and illustrated in FIG. 4.

TABLE 4 Antiviral Activity of the (+) and (−) Enantiomers of FTC %Inhibition Treatment Concn., μM DPM/ml (Corrected) EC₅₀:μM FTC (±)0.0001 73,755 26.6 0.018 0.005 83,005 16.3 0.01 60,465 41.3 0.05 34,12070.4 0.1 14,160 92.4 0.5 18,095 88.1 1 7,555 99.7 5 7,940 99.3 10 5,810101.7 FTC (−) 0.001 76,275 23.8 0.02 0.005 58,590 43.3 0.01 75,350 24.80.05 28,890 76.2 0.1 13,175 93.5 0.5 9,485 97.6 FTC (+) 0.001 94,340 3.80.28 0.005 107,430 −10.6 0.01 99,465 −1.8 0.05 87,120 11.8 0.1 86,34012.7 0.5 33,225 71.4

As indicated in Table 4, the (−)-enantiomer of FTC is approximately oneorder of magnitude more potent than the (+)-FTC enantiomer, and hasapproximately the same anti-HIV activity as the racemic mixture. Neitherthe enantiomers nor the racemic mixture is toxic up to 100 μM asmeasured by Trypan Blue exclusion in human PBM cels.

EXAMPLE 6 Antiviral Activity of Enantiomers Resolved by Method ofExample 2

The enantiomers of (±)-FTC were also resolved by the method of Example2, and the antiviral activity evaluated by the method of Example 4. Theresults are illustrated in FIG. 5. As indicated in FIG. 5, the EC₅₀ ofthe racemic mixture of FTC was measured at 0.017 μM, the EC₅₀ of (−)-FTCat 0.0077 μM, and the EC₅₀ of (+)-FTC at 0.84 μM.

The differences in EC₅₀s as measured in Examples 5 and 6 may be due to anumber of factors, including differences in donor PBM cells, theinherent error of the anti-HIV screening procedure (estimated atapproximately 10%), and differences in the measurement of concentrationof the nucleosides as resolved in the methods of Examples 2 and 3. Inthe method of Example 2, the FTC enantiomers were isolated as solids andweighed to prepare the testing solution. In the method of Example 3, theconcentration of the FTC enantiomers was estimated from UV absorptionmeasurements.

The data indicates that the (+) enantiomer is significantly less potentthan the (−) entantiomer or the racemic mixture.

EXAMPLE 7 Antiviral Activity of the Enantiomers of BCH-189 Resolved byHPLC

The enantiomers of BCH-189 were isolated by the method of Example 3, andthe antiviral activity evaluated by the method of Example 4. The resultsare provided in Table 5, and illustrated in FIG. 6.

TABLE 5 Antiviral Activity of the (+) and (−) Enantiomers of BCH-189 %Inhibition Treatment Concn., μM DPM/ml (Corrected) EC₅₀:μM Blanks mean762 HIV Std. 158,705 Uninfected mean 7,320 Control ±SD 4,520 Infectedmean 97,795 Control ±SD 6,790 BCH-189 (±) 0.001 65,170 36.1 0.081 0.00562,595 38.9 0.01 70,875 29.8 0.05 77,650 22.3 0.1 33,165 71.4 0.5 10,76596.2 1 7,745 99.5 5 6,800 100.6 10 4,470 103.2 BCH-189 (−) 0.001 76,40023.6 0.016 0.005 66,875 34.2 0.01 54,170 48.2 0.05 57,615 44.4 0.134,705 69.7 0.5 15,250 91.2 BCH-189 (+) 0.00085 71,795 28.7 0.23 0.0042599,710 −2.1 0.0085 68,355 32.5 0.0415 82,845 16.5 0.0825 65,100 36.10.412 43,260 60.3

As indicated in Table 6, the (−)-enantiomer of BCH-189 is approximatelyone order of magnitude more potent than the (+)-FTC enantiomer, and hasapproximately the same anti-HIV activity as the racemic mixture. Neitherenantiomer exhibited any toxicity in a concentration up to 100 μM asmeasured by Trypan Blue exclusion in human PBM cells.

EXAMPLE 8 Uptake of (±)-FTC Into Human PBM Cells

Studies were undertaken using radiolabeled agent in order to follow theintracellular profiles of the parent drug and metabolites detectedwithin the cell. All studies were conducted in duplicate. Humanperipheral blood mononuclear cells (PBM cells) are suspended in RPMI1640 medium containing 10% getal calf serum and antibiotics(2×10″cells/ml), 10 ml per timepoint) and incubated with addition of 10μM FTC (specific ctivity about 700 dpm/pmol). Cells are exposed to thedrug for 2, 6, 12 and 24 hours. At these timepoints, the medium isremoved and the cells are washed two times with cold Hank's balancedsalt solution. Extraction is performed with addition of 0.2 ml of 60%cold methanol/water and stored overnight at −70° C. The followingmorning, the suspensions are centrifuged and extractions are repeatedtwo times for 0.5 hours at −70° C. The total supernatants (0.6 ml) arelyophilized to dryness. The residues are resuspended in 250 μl of waterand aliquots comprising between 50 and 100 μl are analyzed by HPLC.Quantitation of intracellular parent drug and metabolic derivatives areconducted by HPLC. Because of the potential acid lability of somecompounds, a buffer system close to physiological pH is used for theseparation of the metabolites.

FIG. 7 is a graph of the uptake of tritiated (±)-FTC in human PBM cells(average of two determinations) in time (hours) versus pmol/10⁶ cells.The uptake studies indicate that radiolabeled FTC is readily taken up inhuman lymphocytes, that produce very large amounts of 5′-triphosphate.

EXAMPLE 9 Uptake of (±)-FTC into Human Liver Cells; HVB Activity of FTC

The same procedure was used with human liver cells as with PBM cells todetermine uptake of FTC.

The (±)-FTC is taken up by hepG2 cells in large amounts. These humanliver cells metabolize a large percentage of the (±)-FTC to (±)-FTCtriphosphate.

These data in conjunction with other data indicate that (±)-FTC, as wellas its (−) and (+) enantiomers, are effective as antiviral agentsagainst HBV (hepatitis B virus).

EXAMPLE 10 Egress of (±)-FTC from Human PBM Cells

Studies were performed using radiolabeled FTC in order to follow theintracellular profiles of the parent drug and metabolites detectedwithin the cell after removal of drug at different times after pulsingfor 24 hours, the time needed for high levels of triphosphates toaccumulate. Studies are conducted in duplicate. Uninfected cells (2-106ml) are suspended in the appropriate medium supplemented with serum (10ml per timepoint) and incubated at 37° C. in a 5% CO₂ incubator.Radiolabeled FTC concentration is 10 μM. After pulsing the cells withthe labeled compound for the desired time, the cells are thoroughlywashed and then replenished with fresh medium without the antiviraldrugs (0 hr). At 0, 2, 4, 6, 12, 24, and 48 hours (second incubationtime), the cells are removed, and immediately extracted with 60% coldmethanol/water. The extract is obtained by centrifugation and removal ofthe cell pellet. The extracts are lyophilized and then stored at −70° C.Prior to analysis, the material is resuspended in 250 τl of HPLC bufferand immediately analyzed. Quantitation of intracellular parent drug andmetabolic derivatives are conducted by HPLC, as follows.

Either a Micromeritics or Hewlett-Packard model 1090 PHLC system is usedwith an anion exchange Partisil 10 SAX column (Whatman, Inc.), at a flowrate of 1 ml/min, 1 kpsi pressure, using uv detection at 262 nm.

The mobile phase consists of:

a. deionized water

b. 2 mM NaH₂PO₄/16 mM NaOAc (pH 6.6)

c. 15 mM NaH₂PO₄/120.2 mM NaOAc (pH 6.6)

d. 100 mM NaH₂PO₄/800 mM NaOAc (pH 6.6)

Separation method: isocratic for 5 min with A, followed by a 15 minlinear gradient to 100% B, followed by a 20 min linear gradient to 100%C, followed by 10 min linear gradient to 100% D, followed by 30 minisocratic with 100% D.

Retention times (minutes) in human cells: Mono- Compound Unchangedphosphate Diphosphate Triphosphate DLS-022 5.0 39.0 55.0 68.0 BCH-1893.5 40.0 55.0 69.0

FIG. 8 is a graph of the egress of radiolabeled (±)-FTC from human PBMcells, measured in hours after drug removal versus concentration(pmol/10⁶ cells). As indicated in the Figure, FTC-triphosphate has anintracellular half-life of approximately 12 hours and can be easilydetected intracellularly at concentrations of 1-5 μM 48 hours after theremoval of the extracellular drug, which is well above the EC₅₀ for thecompound. Further, the affinity (K′) for (±)-FTC triphosphate againstHIV RT is 0.2 μM, below the 48 hour concentration level.

III. Preparation of Pharmaceutical Compositions

Humans suffering from diseases caused by HIV infection can be treated byadministering to the patient an effective amount of (±)-FTC, or its (−)or (+) enantiomer or a pharmaceutically acceptable salt thereof in thepresence of a pharmaceutically acceptable carrier or diluent. The activematerials can be administered by any appropriate route, for example,orally, parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid or solid form.

Pharmaceutically acceptable salts are known to those in the art andinclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acids includehydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric,maleic, phosphoric, glycollic, lactic, salicyclic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic,benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and quaternaryamine.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount of compound to inhibit viralreplication in vivo, especially HIV and HBV replication, without causingserious toxic effects in the patient treated. By “HIV inhibitory amount”is meant an amount of active ingredient sufficient to exert an HIVinhibitory effect as measured by, for example, an assay such as the onesdescribed herein.

A preferred dose of (−) or (±)-FTC will be in the range from about 1 to20 mg/kg of bodyweight per day, more generally 0.1 to about 100 mg perkilogram body weight of the recipient per day.

The compound is conveniently administered in unit dosage form: forexample containing 7 to 7000 mg, preferably 70 to 1400 mg of activeingredient per unit dosage form.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.2 to 70 μM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents. (±)-FTC, or its (−) or (+)-enantiomer orpharmaceutically acceptable salts thereof can be administered as acomponent of an elixir, suspension, syrup, wafer, chewing gum or thelike. A syrup may contain, in addition to the active compounds, sucroseas a sweetening agent and certain preservatives, dyes and colorings andflavors. (±)-FTC, or its (−) or (+)-enantiomers, or pharmaceuticallyacceptable salts thereof can also be mixed with other active materialsthat do not impair the desired action, or with materials that supplementthe desired action, such as antibiotics, antifungals,antiinflamnatories, or other antivirals, including other nucleosideanti-HIV compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

The pharmaceutical composition can also include antifungal agents,chemotherapeutic agents, and other antiviral agents such as interferon,including α, β, and gamma interferon.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, aractiadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives are thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperse.lipid aggregates, thereby forming the liposomal suspension.

IV. Preparation of Phosphate Derivatives of FTC

Mono, di, and triphosphate derivative of FTC can be prepared asdescribed below.

The monophosphate can be prepared according to the procedure of Imai etal., J. Org. Chem., 34(6), 1547-1550 (June 1969). For example, about 100mg of FTC and about 280 μl of phosphoryl chloride are reacted withstirring in about 8 ml of dry ethyl acetate at about 0° C. for aboutfour hours. The reaction is quenched with ice. The aqueous phase ispurified on an activated charcoal column, eluting with 5% ammoniumhydroxide in a 1:1 mixture of ethanol and water. Evaporation of theeluant gives ammonium FTC-5′-monophosphate.

The diphosphate can be prepared according to the procedure of Davissonet al., J. Orq. Chem., 52(9), 1794-1801 (1987). FTC diphosphate can beprepared from the corresponding tosylate, that can be prepared, forexample, by reacting the nucleoside with tosyl chloride in pyridine atroom temperature for about 24 hours, working up the product in the usualmanner (e.g., by washing, drying, and crystallizing it).

The triphosphate can be prepared according to the procedure of Hoard etal., J. Am. Chem. Soc., 87(8), 1785-1788 (1965). For FTC is activated(by making a imidazolide, according to methods known to those skilled inthe art) and treating with tributyl ammonium pyrophosphate in DMF. Thereaction gives primarily the triphosphate of the nucleoside, with someunreacted monophosphate and some diphosphate. Purification by anionexchange chromatography of a DEAE column is followed by isolation of thetriphosphate, e.g., as the tetrasodium salt.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, a method ofresolution and antiviral activity of nucleoside enantiomers, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of theappended claims.

We claim:
 1. The (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-onethat is at least 95% free of the corresponding (+)-enantiomer. 2.(−)-Cis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt, ester or salt of an esterthereof.
 3. The substantially pure (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt, ester, or salt of an esterthereof, wherein the (+) enantiomer is present in an amount of no morethan 5% w/w.
 4. The compound of claim 3 wherein the (+)-enantiomer ispresent in an amount of no more than about 2% w/w.
 5. The compound ofclaim 3 wherein the (+)-enantiomer is present in an amount of less than1% w/w.
 6. A pharmaceutical composition comprising a compound as claimedin any one of claims 2-5 in combination with a pharmaceuticallyacceptable carrier. 7.(−)-Cis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt thereof.
 8. The 5′-O-alkylderivative of the (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-one.9. The 5′-O-alkylC(O)-derivative of the (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-one.10. The derivative of claim 9, wherein alkylC(O)— is selected from thegroup consisting of acetic, propionic, butyric, and pentanoic.
 11. Themonophosphate, diphosphate, or triphosphate of the (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-one.12. A pharmaceutically acceptable salt of the (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-onethat is at least 95% free of the corresponding (+)-enantiomer.
 13. Apharmaceutical composition comprising an effective HIV treatment amountfor humans of the (−)-enantiomer ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-onethat is at least 95% free of the corresponding (+)-enantiomer, incombination with a pharmaceutically acceptable carrier or diluent.
 14. Apharmaceutical composition comprising an effective HIV treatment amountfor humans of the (−)-enantiomer of a pharmaceutically acceptable saltof a compound ofcis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimdin-2-onethat is at least 95% free of the corresponding (+)-enantiomer, incombination with a pharmaceutically acceptable carrier or diluent. 15.The pharmaceutical composition of claim 13, in a form for oraladministration.
 16. The pharmaceutical composition of claim 15, whereinthe composition is in tablet form.
 17. The pharmaceutical composition ofclaim 15, wherein the composition is in capsule form.
 18. Thepharmaceutical composition of claim 13, wherein the composition is aliquid.
 19. The pharmaceutical composition of claim 13, in a form forintravenous administration.
 20. The pharmaceutical composition of claim19, wherein the carrier comprises a sterile diluent for injection. 21.The pharmaceutical composition of claim 13, in a form for topicaladministration.
 22. The pharmaceutical composition of claim 14, in aform for oral administration.
 23. The pharmaceutical composition ofclaim 22, wherein the composition is in tablet form.
 24. Thepharmaceutical composition of claim 22, wherein the composition is incapsule form.
 25. The pharmaceutical composition of claim 14, whereinthe composition is a liquid.
 26. The pharmaceutical composition of claim14, in a form for intravenous administration.
 27. The pharmaceuticalcomposition of claim 17, wherein the carrier comprises a sterile diluentfor injection.
 28. The pharmaceutical composition of claim 14, in a formfor topical administration.